This inventions relates to pharmacologically-active, anti-inflammatory compounds, and more specifically to long aliphatic chain esters of selected zwitterionic organic compounds derived from naturally occurring taurine. This invention pertains to novel zwitterionic compounds with pharmacologic activity including, but not limited to, their use as an anti-inflammatory agents.
Any inflammation that occurs in the mammalian body is the clinical result of a sequence of events known as the arachidonic acid (ARA) cascade. Cell membranes consist of phospholipids, including fatty acids, one of which is ARA. In the inflammation process, the first step is the release of ARA from the phospholipid. The next step is the conversion of ARA into the specific mediator of inflammation. One pathway is the cyclooxygenase enzyme, and the other is called the lipoxygenase pathway. Cortisone, along with other selected steroidal agents, block both inflammation pathways by inhibiting ARA release from the phospholipids.
The mode of action of HEPES-ester is thought to be at the level of leukotrienes B4, but it is also possible that it occurs at higher levels in the inflammatory cascade, perhaps at the phospholipase A2 (PPLA2). Successful Inhibition of PPLA2 action would arrest the aforedescribed cascade effect from being initiated.
Medical science searches for other biochemicals that lack the recognized side effects of prolonged steroid-based (hydrocortisone) medications. One known human biochemical, taurine (2-amino ethanesulfonic acid), synthesis of which occurs in the mammalian liver, has demonstrated anti-inflammatory activity when administered centrally, but not when administered subcutaneously or interperitoneally. N-substituted derivatives of taurine include: 4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid; C8H18N2O4S, which derivatives are commonly identified in the technical literature as HEPES (Merck Index, 12th edition monograph #4687). HEPES itself is available commercially from Angus Chemicals, as the sodium salt or, as the free acid. The scientific literature reports that intravenous injection of (14-C) HEPES, or of (3H) taurine, demonstrated rapid clearance, but with a significantly longer half-life compared with taurine. Mahon et al theorized that the greater anti-inflammatory effects of HEPES (sodium salt and the acid), as compared with taurine, may be due to its slower systemic distribution or clearance, in vivo. The prior art suggest that HEPES is a significant agent to reduce cellular inflammation and cellular proliferation. However, the safe delivery systems for the HEPES treatment of inflammation remain to be optimized.
It is thus a principal object of this invention to provide a HEPES-based compound, an ester, and a pharmaceutically acceptable formulation including the ester, that is adapted for use in topically applied products so as to reduce symptoms of skin inflammation, wherein the particular etiology of the inflammation does not call for, or require, the use of antibiotics or germicidal compositions. Improved formulations for epidermal penetration, on bruises, muscle strains and sprains are also areas of useful progressive treatment.
It is another object of the invention to complex the HEPES molecule with selected aliphatic acids, such serving as the active ingredient of topical applications, which permit the HEPES moiety to penetrate the skin and so to better effect its anti-inflammatory nature.
It is a further object of the invention to provide a HEPES-containing active ingredient that is not limited to the known subcutaneous injection or IV infusion routes, but may also effective as a topical formulation.
A still further object of the invention is to provide HEPES esters as a cosmetic formulation ingredient, as a co-emulsifier, usable with topical analgesics.
Still another object of the invention is in a cosmetic preparation to incorporate an anti-skin ageing active ingredient.
These and other objects and benefits of this invention will become apparent from a study of the following specification.
The present invention relates to a composition and method for the treatment of inflammatory conditions in mammals, by the topical administration of selected Zwitterionic ester compositions, serving as safe and effective substances. Among useful Zwitterionic compounds which are presently preferred; these include EPES, PIPES, BES, POPSO, and most preferably HEPES, when esterified, then alone, or in combination with other therapeutic ingredients. They are employed by applying to an affected area of the skin, a therapeutically effective amount of at least one skin compatible, Zwitterionic-Ester having the generic formula: 
wherein M is an alkali metal and R is a naturally occurring, straight-chain, saturated or unsaturated, aliphatic acid moiety selected from one of the hydrocarbon groups consisting of alkanes, alkenes, and alkadienes, each group having a chain of from one to twenty carbon atoms. The resulting HEPES esters are represented by the acetic acid ester as an exemplary alkane, the oleic acid ester as an exemplary alkene, and the linoleic acid ester as an exemplary alkadiene, the esters of which do form fatty acids in nature.
Preferably, a Zwitterionic-ester has at least one pKa value at 20xc2x0 C. in the range of 6.0-8.3, to permit its use on human skin, i.e., the ester exists mainly in its dipolar form, in the pH range of 6.0-8.3.
The isoelectric (ISO) point is the pH at which the net charge on a molecule in solution is zero. At this pH, amino acids exist almost entirely in the Zwitterionic state, i.e., the positive and negative groups are equally ionized. A solution of amino acids at the ISO point exhibits minimum conductivity, osmotic pressures, and viscosity.
Such dipolar molecules contain, for example, hydroxy groups and amino groups, and also acid groups, like phosphoric, carboxylic, or sulfonics acid groups and, generally have pKa""s in the range of 6.15-8.4.
Preferred aliphatic values for the ester moiety of the HEPES ester are n-butanoic; isobutyric, n-valeric, palmitic, and stearic, behenyl, lauric, myristic, (and their isomers), among the saturated aliphatics; and oleic and linoleic, among the unsaturated aliphatics.
The invention also provides a pharmaceutical composition for application to human skin in the treatment of inflammation comprising at least one of the above Zwitteronic-esters as the active ingredient, together with a pharmacologically acceptable topical carrier or base.
These select esters may be useful in the treatment of arthritis, myositis, insect bites, sunburn, psoriasis, atopic dermatitis, and other inflammatory processes of muscle, connective tissue, or skin appendages.
The effective proportion of the active ester ingredient, by weight of the formulation, is in the range of 1 to 20%, preferably 5 to 10%. In the most preferred composition, the effective proportion lies in the range of 6 to 8%. Not every compound falling within the general definition given above, is suitable for topical use per the method of the invention. Some few will prove to be contraindicated. Nevertheless, the exclusion of ineffective active ingredients is a matter well within the competence of the skilled pharmacologist in the conduct of the anti-inflammatory evaluation protocols (to be described) for disclosed HEPES-esters.
The topical formulation base is selected from a wide variety of compositions, formulated according to known principals for pharmaceutical purposes. Such compositions include creams, solids, ointments, lotions, and film-forming solutions among others. They may be presented in boxes, jars, or compressible tubes, both collapsible and non-collapsible. The solids may be presented as sticks for rubbing onto the skin. Some of the topical bases may be presented as papers, woven or non-woven fabric pieces, or pads, all being impregnated with composition.
The invention relates to HEPES derivatives which are pharmacologically active as anti-phospholipase and anti-inflammatory compounds specifically, wherein the active ingredients are certain long chain esters of selected zwitterionic compounds, based on an N-substituted taurine, namely aliphatic esters of HEPES.
The novel compounds may exist as at least one of the following five organic groups: ester, ether, urethane, amide, or urea of all of the following known compounds, and their salt forms. Preferred Zwitterionic-esters are prepared from the below listed sulfonic acids.
ACES-N-(2-Acetamido)-2 amino ethane sulfonic acid.
AMPSO-3-[1,1Dimethyl-2 hydroxyethyl amino]-2-hydroxypropane sulfonic acid.
BES-N,N-bis (Hydroxyethyl)-2 aminoethane sulfonic acid.
DIPSO-3-[N,N-bis (hydroxyl ethyl)amino]-2-hydroxypropane sulfonic acid.
CAPS-3-(cyclohexylamino)-1-propane sulfonic acid.
CAPSO-3-(cyclohexylamino)-1-propane sulfonic acid.
CHES-2-(N-cyclohexylamino)ethane sulfonic acid.
HEPPS-N-(2-hydroxy ethyl)piperazine-Nxe2x80x2-3-propane sulfonic acid.
HEPES-N-(2-hydroxy ethyl)piperazine-Nxe2x80x2-(2-ethane sulfonic acid).
HEPPSO N-(2-hydroxy ethyl)piperazine-Nxe2x80x2-2-hydroxypropane sulfonic acid.
MES-2-(-Morpholino)ethane sulfonic acid.
MOPS-3-(N-Morpholino)propane sulfonic acid.
MOPSO-3-(N-Morpholino)-2 hydroxy propane sulfonic acid.
PIPES-Piperazine-N, Nxe2x80x2-bis (2 ethane sulfonic acid).
New Mono PIPES-Piperazine-Nxe2x80x2-(2 ethane sulfonic acid).
POPSO-Piperazine-N, Nxe2x80x2-bis (2-hydroxy propane sulfonic acid).
TAPS-3-[N-tris-(hydroxy methyl)methyl amine]-2-hydroxy propane sulfonic acid.
TES-N-tris-(hydroxy methyl)-methyl-2-amine ethane sulfonic acid.
EPES-N-ethylpiperazine-N-(2 ethane sulfonic acid).
EPPS-N-ethylpiperazine-N-(2 propane sulfonic acid).
The precursor compounds listed above are best known in the literature as biological buffers. Many are commonly used as buffering agents in mammalian cell cultures. Manufacturers of these compounds include Angus Chemicals, SIGMA, and British Drug House.
Synthesis of HEPES Esters
Generally, an alkali metal salt of HEPES is catalytically reacted with an alkyl-substituted, either saturated or unsaturated, aliphatic salt, such as methyl oleate, methyl linoleate, methyl palmitate, methyl stearate, methyl myristate, and methyl behenate. They are reacted in equi-molecular amounts, carried out either with or without a non-aqueous solvent, such as acetone, and in a temperature range of 0xc2x0 C. to variable xc2x0 C., which is between 0xc2x0 C. and the chosen solvent""s reflux temperature. The purification of the crude ester is carried out by means of crystallization in an organic solvent, dissolved in methanol, and recrystallized. As to analytical methodology, preparative high pressure liquid chromatography (HPLC) is followed by TLC, HPLC, and nuclear magnetic resonance (NMR) techniques. Potentiometric titration and bromic titration are done across the double bond to establish identity and purity. The HPLC device is equipped with a variable length, ultraviolet detector.
Sodium lauryl sulfate solution 0.5% is applied in a patch to four areas on the volar surface forearm. Site one is pretreated with a base formula, site two is pretreated with the base formula plus 10% HEPES ester, while skin sites three and four are left untreated.
After 24 hours, the sites are examined, the erythema and edema on sites one and two are recorded by photographs. Site one is treated with the base product, site three is treated with the base product plus 10% HEPES ester, and sites two and four are untreated controls. Each treated site (two) is treated four times a day. The observed results are recorded as the percent reduction of erythema and edema at the HEPES ester-treated sites over the base formula site and two control treated sites.

The preparation of Compound I is characterized by catalytically reacting 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, with salts like methyl oleate, C17H33CO2CH3, in equimolar amounts. The reaction can be carried out in a non-aqueaous solvent. Sodium methoxide is a useful catalyst. The reaction temperature may vary between 0xc2x0 C. and the solvent reflux temperature. The purification of the compound is carried out by means of crystallization in a solvent such as acetone.
I. The Esters
Methyl oleate HEPES Na+Na methoxidexe2x86x92HEPES oleate+Methyl alcohol HEPES Na oleate 

The preparation of Compound I can be carried out by reacting a fatty acid with HEPES (full compound name) or any one of its salt derivatives. One preferred process route is to react HEPES, sodium salt, with methyl oleate and sodium methoxide as a catalyst. 
The reactions is carried out with an excess of methyl oleate as a solvent. To ensure the completion of the reaction, removal of side product methanol is carried out.
Preparation of the HEPES oleate: sodium salt 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, sodium salt, is slowly added to a stirring flask of methyl oleate in equal molar equivalents. The mixture is stirred at room temperature for 15 minutes. Then a catalytic quantity of sodium methoxide is slowly added to the stirring mixture. The reaction mix is stirred for less than five minutes. The reaction mix is then slurried in acetone, then isolated and dried, affording greater than an 80% yield of the title oleate as a white to off-white solid, to wit: 